Production of soy sauce

ABSTRACT

Soy sauce is produced in a relatively short time by fermenting Maromi with an inoculant selected from Saccharomyces and Debaryomyces or a Maromi isolate.

United States Patent Luksas et al.

[ Apr. 15, 1975 Related US. Application Data Division of Ser. No.36.427, May 11, 1970, Pat. No. 3,711,303.

US. Cl. 426/46; 426/60; 426/62;

426/203; 426/204; 426/221 Int. Cl A231 1/20 Field of Search 99/145;426/46 References Cited UNITED STATES PATENTS 2/1970 Mogi et a1 99/1453,552,981 1/1971 Luksas 99/145 FORElGN PATENTS OR APPLlCATIONS 3,5426/1962 Japan 99/145 Primary Examiner'Samih N. Zaharna AssistantExaminer-J. M. Hunter Attorney, Agent, or FirmCushman, Darby and Cushman[57] ABSTRACT Soy sauce is produced in a relatively short time byfermenting Maromi with an inoculant selected from Saccharomyces andDebaryomyces or a Maromi isolate.

17 Claims, No Drawings PRODUCTION OF SOY SAUCE This is a division. ofapplication Ser. No. 36.427 filed May ll. 1970, now US. Pat. No. 3,71L303.

The present invention relates to the production of soy sauce and to soysauce having the natural flavor and taste'of aged soy sauce. Theinvention further relates to such soy sauce which may be produced in arelatively short time.

Historical records indicate that the Chinese have been using soybeanproducts for well over 3.000 years. A wide variety of foods have beendeveloped by the Chinese based on soybeans as a principal ingredient.Soy sauce, one of the older Chinese products. has become an importantcondiment in the United States. not only for its use as a flavoringagent alone, but as an ingredient for mixing with other flavoring agentsto produce additional sauces and the like. e.g., Worcestershire sauce.which contain from to percent soy sauce. Soy sauce may be manufacturedby two main processes.

Soy sauce may be made by fermentation or enzymatic hydrolysis of proteinto give amino acids and lowmolecular-weight polypeptides or by the acidhydrolysis process. The former of these two processes. i.e.. thefermentation process, is by far the more important process, since itgives the soy sauce a full body and flavor as opposed to the acidhydrolysis process. Hence, most of the soy sauce, especially the higherquality Oriental type soy sauce, manufactured in the United States ismade by the fermentation process.

Briefly stated, the fermentation process for preparing soy saucecomprises preparing a mixture of washed, cooked and drained soybeanswith parched wheat, or other grains or cereals, and adding to themixture (without additional water being used) a Koji culture. The Kojiculture is a naturally produced culture, and the exact composition ofKoji culture is not fully known. However, generally speaking Kojiculture contains mold, yeast and lactic acid bacteria. The mixture ofsoybeans, parched wheat and the Koji culture is allowed to biologicallyalter for 4 to 10 days and this biologically altered mixture is calledKoji. The Koji is added to an aqueous salt solution. The mixture ofaqueous salt solution and Koji is called Maromi. The Maromi is thenallowed to ferment for an extended time period. While some soy sauceflavor can be developed after fermentation of the Maromi for as littleas 30 days, for full-bodied and full-flavored soy sauce, thefermentation is normally allowed to proceed for at least six months andpreferably up to a year or more, e.g., 18 months. After the fermentationis complete and the desired degree of body and flavor has been reached,the fermented mass is pressed and the liquid obtained therefrom is soysauce. The soy sauce may be further mixed with other agents such assalt. coloring, preservatives and the like. As can be noted from theabove discussion of the fermentation process for producing soy sauce,the fermentation step requires that the Maromi be allowed to ferment foran extended period of time and that large holding vessels for fermentingthe mixture are necessary to produce sizable amounts of soy sauce. Ascan be also appreciated, it would be desirable to shorten the time ofthe Maromi fermentation step required to produce a full-bodied andfull-flavored soy sauce. Equally important, however, is the requirementthat any shortened fermentation time of the Maromi must still result ina full-flavored and fullbodied soy sauce, of the quality expected by thegeneral consuming public.

Therefore it is an object of this invention to provide a process forpreparing a full-bodied and full-flavored soy sauce with a shortenedMaromi fermentation time. lt is a further object of the invention toprovide a soy sauce of the fermented type which is full-flavored andfull-bodied but which has been produced from Maromi that was fermentedin a relatively short period of time. Other objects of this inventionwill be apparent from the following disclosure and claims.

Basic to the present invention is the discovery that certain organismswill develop the characteristic soy sauce flavor in fermenting Maromi.Having discovered these flavor producing organisms, a process has beendevised wherein these organisms are grown during the fermentation ofMaromi at a very rapid rate to produce the characteristic soy sauceflavor in a relatively short fermentation time.

In order to completely appreciate the significance of the presentinvention, reference is made in detail to the conventional process forproducing a fermented soy sauce. The conventional process is generallyreferred to in the art as the Koji and Maromi" process. The Koji cultureis essentially a starter inoculant. the total composition of which isnot fully known to the art, as noted above. In a general sense, Kojiculture is simply a biologically active portion of a Koji and which maybe optionally concentrated and dried. The origin of the first Kojiculture is lost in antiquity and in the Koji and Maromi process it isnecessary to have a Koji culture in order to produce additional soysauce. Koji culture can be obtained on the open market. and theprincipal sources of Koji culture are Japan and China, especially Japan.Koji culture may be obtained in a liquid form as well as the dry form.noted above, and may be mixed with prepared soybeans and wheat toproduce additional Koji culture or produce Koji from which soy sauce ismade by fermentation. In other words, Koji culture is a starter cultureof not fully known composition which has been handed down fromgeneration to generation for use in producing soy sauce via the Koji andMaromi" process.

ln the preferred process in the art, an equal mixture of roastedsoybeans and boiled soybeans (with excess liquid removed) is mixed withan equal amount of roasted wheat. After thorough mixing of the roastedand boiled soybeans with the roasted wheat, the mixture is placed in asuitable vessel and inoculated with Koji culture. The Koji culture maybe concentrated and powdered Koji culture which is commerciallyavailable or the Koji culture may be simply a biologically active partof a previously produced Koji. Generally from 1 to 10 percent by weightof the soybean, wheat, etc. of the Koji culture, which is obtained froma previously produced Koji, is used as the inoculant in the mixture.When concentrated powdered Koji culture is used as the inoculant, theamount thereof may vary between 1 to parts by weight of powdered Kojiculture to 1,000 to 200,000 parts by weight of soybean, wheat. etc. Thisaddition of Koji culture, either in concentrated, powdered form or froma previously produced Koji is well known in the art and is conventional.Hence, any conventional amounts may be used. ln either case, theinoculated mixture is fermented for 3 to 10 days, preferably about 5days, at 60 to l 10F, especially about 68 to 90F. Generally speaking thetemperature of fermentation will be between 80 and 84F. During thisperiod the Koji culture produces growth mainly of largely unidentifiedmolds, yeasts and bacteria in the mixture of soybeans and wheat. (Thismixture of soybeans, wheat and growing Koji culture organisms isreferred to as Koji.) After this initial fermentation period, themixture of soybeans and wheat with the Koji culture organisms growingtherein (now referred to as Koji) is placed in an 18 w/v percent aqueoussalt solution. Generally from 5 to 40 w/v percent of the Koji is placedin the salt solution. This mixture of the Koji and salt solution isreferred to as Maromi. The Maromi is mixed in a large tank to obtain aneven distribution. Thereafter mixing may or may not be performed,depending upon the particular manufacturer making the soy sauce. Mixingof the Maromi is, however, not necessary. The Maromi is then allowed toferment at 60 to 110F, especially 68 to 90F and ideally 80 to 84F for anextended period of time. While this extended period of time may varywidely depending upon the flavor, body and bouquet desired, good qualitysoy sauce is only produced with fermentations which are allowed toproceed for at lesat 9 months and the better fullflavored soy sauce isallowed to ferment for as long as 18 months, especially the flavorassociated with Oriental style soy sauce. At the end of the fermentationperiod, the liquid associated with the Maromi is separated from thesolids. The solids are discarded and the liquid is further processed,e.g., pasteurized and compounded with other ingredients to produce soysauce.

Broadly stated, the present process for producing soy sauce comprisesfermenting conventional Koji, as described above, in the presence ofspecific organisms, as hereinafter defined, and in a salt solution withmixing under an inert gas blanket with short periods of aeration.continuing the mixing and fermenting with intermittent aeration for upto 5 weeks and pressing the resulting fermented mixture to obtain afull-flavored, fullbodied, Oriental style soy sauce.

It is greatly preferred that the Koji be comminuted to a relativelysmall particle size, e.g., by grinding. After grinding, all particlesshould pass through a number 3 US sieve screen, but much improvedresults are obtained when all of the particles will pass through atleast a number 6 US sieve screen, and especially the average particlesize should be less than 1,000 microns, e.g., l to 100 microns. Anyconventional type grinder or comminuting apparatus may be used forcommuting the Koji. for example, an Urschel mill, a gear grinder, aRietz mill or a stone grinder. However, other grinders such as ballgrinders, rod grinders, hammer mills, etc., may be used. It is onlynecessary that the particle size after grinding be within the rangenoted above. However, it is most important that during grinding thetemperature of the Koji be kept below 145F, especially below 130F, e.g.below 100F, in order to prevent the destruction of the enzymes containedin the Koji. Salt may be added to the Koji either before or aftergrinding. Conveniently the salt is added to the Koji prior to grindingso that the salt may be ground to a very small particle size during thegrinding process. Alternately, instead of grinding the Koji, thesoybeans, wheat, etc., from which the Koji is made may be ground priorto making the Koji, in the same manner as described above. In this case,the above noted temperature requirements are not so critical.

The Koji is then added to water and at this point salt is added, if thesalt was not added during the grinding process. The resulting mixture ofKoji, salt and water (called Maromi) should contain from 5 to 40 w/vpercent of the Koji. especially between 20 and 25 w/v percent. Theamount of salt in the resulting mixture, added either prior to grindingor after grinding, should be between about 6 to 30 w/v percent,especially between 15 and 20 and ideally about 18 w/v percent. Whilecommon sodium chloride is the preferred salt. especially in view of therelatively low cost thereof, other salts may be used, e.g., halides,especially the chlorides, of alkaline earth and alkali metals orammonia. in any event the salt must be an acid salt and particularlysuitable are sodium and potassium chloride, calcium chloride andmagnesium chloride. Organic salts may also be used, if desired, e.g.,sodium acetate and sodium lactate. Nevertheless sodium chloride is thepreferred salt and will normally be used in the process.

The mixture of Koji in the aqueous medium (hereafter referred to asMaromi) is then agitated and to the agitated Maromi is introduced aninoculant, as hereinafter further defined.

After inoculation, the Maromi is initially fermented for from 3 to 14days. Of course longer periods than l4 days may be used but there is nosubstantial advantage in continuing the fermentation longerthan 14 days,and generally no advantage is obtained in continuing the fermentationbeyond 7 days. The initial fermentation temperature will be between 60and llOF and especially between 68 and 90F, e.g. and 84F. The initialfermentation is carried out in a closed vessel having an inert gas headspace. But preferably, at least some intermittent aeration is carriedout during the initial fermentation. The inert gas provides essentiallyanaerobic conditions and the anaerobic conditions may be insured bycontinuously or intermittently flushing with the inert gas. Theintermittent aeration may be carried out by flushing the head space ofthe vessel with an inert gas, such as nitrogen, containing low levels ofoxyshort periods of time. This may be accomplished by stirring thefermenting Maromi and sweeping oxygencontaining gases, e.g., air,through the head space of the closed vessel. The amount of oxygen in thehead space of the closed vessel during these aerations may be quite low;similarly the dwell period to the oxygen containing gas may be quiteshort, and can vary from as quickly as the oxygen containing gas canreplace the inert gas in the head space and the oxygen containing gascan be subsequently replaced by the inert gas to up to 2 or 3 hours ormore. It is not necessary that the oxygen containing gas be allowed todwell in the head space for any extended period of time and only amomentary displacement of theinert gas in the head space is necessary.However, generally speaking, the oxygencontaining gas will be allowed to.dwell in the head space from 1 to 50 minutes, especially from 4 to l5minutes and preferably about 5 minutes. The frequency if aeration canvary widely and may be from as little as once every four days to asoften as once every l() minutes, especially from once every 2 days to 5times a day and preferably approximately 2 to 3 times a day. Of course,during this aeration period the fermenting Maromi should be agitated.

The purpose of these intermittent aeration steps is to essentiallyaccomplish in the fermenting Maromi a microaerophillic condition. i.e..a state of oxygen tension that is less than atmospheric and being on theborder between aerobic and anaerobic conditions. As can be appreciatedfrom the above. the initial fermentation can be under either anaerobicor microaerophillic conditions, but microaerophillic conditions aregreatly preferred. It has been found that microaerophillic conditions,at least for part of the fermentation time, develop a superiorfull-flavor and full-body soy sauce according to the present invention.

Alternately, instead of intermittent aerations, as discussed above,microaerophillic conditions may be established on a continuous basis byemitting with the inert gas an extremely low level of oxygen into thehead space of the closed vessel. The amount of oxygen introduced withthe inert gas into the head space of the closed vessel will vary widelydepending upon the volume of the head space, the volume of thefermenting mixture, the temperature of fermentation, the state offermentation, etc. Hence no specific amounts of oxygen can be stated asa general case, but one skilled in the art can readily determine whenmicroaerophillic conditions are produced within the fermenting Maromiand adjust the oxygen content to maintain those microaerophillicconditions. However, when the process is carried out using continues lowlevels of oxygen in the gases flushing the head space, it is necessaryto continually determine that microaerophillic conditions are beingmaintained in the fermentation process. This of course requires aconsiderable amount of effort in following the fermentation process andfor this reason it is preferred to intermittently aerate the fermentingMaromi, as noted above, rather than attempting to carry out thefermentation with continuous but low levels of oxygen in the inert gas.

After the initial fermentation period, preferably with aeration asdescribed above, the fermenting Maromi is further fermented for anadditional 5 to days or longer, if desired, either in a sealed vessel orwith low levels of intermittent or continuous aeration. Hence, thesecond fermentation may be anaerobic, microaerophillic or mildlyaerobic, but microaerophillic or mildly aerobic conditions are greatlypreferred, since these conditions produce a superior ultimate flavor.Preferably the Maromi is agitated during the aerations.

It should also be clearly understood that while the best flavor, bodyand bouquet are developed according to the present process when aerationis carried out in the second fermentation step, an acceptable, butinferior flavor, bouquet and body may also be obtained without anyaeration during the subsequent fermentation step and wherein thesubsequent fermentation step is carried out under an inert atmosphere.However, since superior flavor, body and bouquet are developed when thefermentation is carried out with the above described aerations in thesecond fermentation step, this is the preferred embodiment of theinvention.

The second fermentation may be carried out at the same temperatures asthe initialfermentation and indeed the same procedure for introducingoxygen into the head space may be used. Hence, the second fermentationstep may be simply an extension of the first fermentation step,especially where microaerophillic conditions are used in both the firstand second fermentations.

After the subsequent second fermentation step. the solids are separatedfrom the liquid by any conventional means. e.g,, filtering, pressing.centrifuging. vacuum distillation, etc. The recovered liquid is thenmixed with conventional additives such as salts. edible coloring, e.g.,caramel, other flavors. preservatives.

sweeteners. such as sugars. spices. etc. to produce the finished soysauce ready for sale to the consuming public. Alternately, the productmay be pasteurized either before or after the addition of the additivesor before packaging. It is not necessary, however, to pasteurize the soysauce and indeed some additional flavor will develop after packaging thesoy sauce when the soy sauce has not been pasteurized.

The inoculant used with the present process is a yeast of the familyEndomycetaceae, subfamily Sac charomycetoideae, tribe Saccharomyceteae,and particularly useful yeasts ofthe said tribe are selected fromSaccharomyces and Debaryomyces.

For example, species and varieties which may be used includeSaccharomyces cerevisiae, Saccharomyces cerevisiae var. ellipsoideus,Saccharomyces pastorianus, Saccharomyces rouxii. Saccharomyces rouxiivar. polymorphus, Saccharomyces exiguus, Saccharomyces marxianus,Saccharomyces bailii, Saccharomyces logos v. Laer et Denamur,Saccharomyces bayanus, Saccharomyces willianus, Saccharomyces uvarum,Saccharomyces delbrueckii, Saccharomyces delbrueckii, var. mongolicus,Saccharomyces carsbergensis, Saccharomyces fragilis, Saccharomyceslactis, Saccharomyces rosei, Saccharomyces chevalieri, Saccharomycesbisporus, Saccharomyces pastori, Saccharomyces fermentati, Saccharomycesheterogenicus, Saccharomyces microellipsodes, Saccharomyces oviformis,Saccharomyces mellis, Saccharomyces italicus, Saccharomyces florentinus,Saccharomyces acidifaciens, Saccharomyces steineri, Saccharomycesfructuum, Saccharomyces elegans, and Saccharomyces veronae. All of thespecies and varieties are known to the art and are easily obtainable.

The particular species of Debaryomyces which may be used includeDebaryomyces hansenii, Debaryomyces kloeckeri, Debaryomyces subglobosus,Debaryomyces vini, and Debaryomyces nicotianae. Likewise the species areknown to the art and are readily obtainable.

For a complete discussion of the above-noted yeasts see The Yeasts,Lodder, J., et a1, North-Holland Publishing Company, Amsterdam, 1967,especially pages 1 l7l22 and 270-279, which disclosure is incorporatedherein by reference. The foregoing reference gives the key toDebaryomyces and Saccharomyces I for identification and isolationpurposes.

However, in a preferred embodiment of the invention the inoculant isisolated from Maromi since the isolated inoculant gives a better flavorto the soy sauce as opposed to the flavor obtained by using commerciallyavailable organisms. The isolated inoculant can be obtained by thefollowing procedure. Prepare two aqueous solutions of YM agar powder (acommercially available material and fully identified hereinafter), one

with 18 w/v percent of NaCl and one with water only (approximately gramsof YM agar powder per 100 ml. of aqueous NaCl or water). Stcrilize thesolutions. e.g. at 250F for minutes, and prepare plates by placing l5 to20 cc. of each solution in separate sterilized petri dishes and cool toll3l2()F. lnoculate each plate with approximately 0.1 to 0.3 cc. of oneyear old Maromi (produced by the conventional process as noted above) bystreaking out with a bacteriological needle. as is the common practicein the art. lncubate the inoculated plates for about 4 days at 71F (somegrowth will be noted after 48 hours). After incubation. with abacteriological needle. remove the yeast growths from both the water andsaline plates (one skilled in the art can easily identify yeast growthsfrom bacteria and mold growths by visual observation) and place eachdifferent yeast growth on a separate YM agar plate prepared or YM agarplate with NaCl, as discussed above. Of course, yeasts taken from YMagar plus salt plates are placed on new YM agar plus salt plates andyeasts taken from YM agar plates are placed on new YM agar plates.Serially grow the yeasts on new YM agar plates and YM agar plus salt.respectively, as discussed above, and separate the yeast from each plateand grow on new plates again until essentially pure cultures of yeastsare obtained and the cultures are essentially free from bacteria andmold cultures. Of course, the serial growing of the yeasts isaccomplished at the above temperatures and times. The number of serialgrowings of the yeasts will, of course, depend upon how carefully theyeasts are removed from the plates.

The above procedure is simply that for obtaining pure cultures by serialgrowths and is quite standard in the art and one skilled in the art willhave absolutely no trouble in obtaining pure yeast cultures.

After all of the yeast cultures have been purified, as discussed above,the purified cultures are visually observed under daylight and comparedwith respect to the following appearances: dull, shiny (glistening) andflaky. The observer will see that some of the yeast cultures arestrikingly different from other of the yeast cultures in these threerespects, and dull, shiny and flaky cultures will be readily apparent.(With proper streaking ofthe first Maromi culture, these threeappearances will be readily apparent in the first culture of the Maromiand by retaining only the cultures having these three appearances thenumber of cultures and number of serial growing may be materiallyreduced.)

The cultures having a dull, shiny or flaky appearance will be thosecultures containing the organisms of the present invention. Any one ofthese cultures may be used to produce soy sauce. but a combination ofthe three cultures produce an exceptional flavor, body and bouquet.

An alternate procedure for obtaining the Maromi isolated organisms" maybe used. The procedure is follows. lnto distilled water is placed 4 w/vpercent of agar and cooked at boiling to dissolve the agar. Thedissolved agar solution is autoclaved and cooled to room temperature, orat least below 45C. Several plates are made from the agar by mixing theagar with an equal volume of naturally produced soy sauce (produced bythe conventional Koji and Maromi process). The soy sauce used in mixingwith the agar should be essentially pure soy sauce and suitable purityis obtained by passing the soy sauce through a bacteriological filter.After the mixture of soy sauce in agar solution has solidified in theplates, the plates are inoculated with aged Maromi obtained from aconventional Koji and Maromi process by streaking the plates with abacteriological needle. The Maromi used for streaking the plates shouldbe at least one month old Maromi but Maromi of twelve months old orlonger is desired. The plates are then placed in a conventionalanaerobic jar or any other like device to obtain anaerobic conditions.The plates are incubated at mild temperatures, e.g., up to 40C,especially at room temperature, for 2 to 4 months under anaerobicconditions. After about 1 month and especially after about two months.distinct colonies of organisms will be observed on the plates. Therewill be three very distinct and easily recognizable different coloniesand essentially no other colonies of any appreciable size will becontained on the plates. These three distinct colonies. by visualobservation. will be characterized in that one will be dry. flaky.white, large and rough; the second will be large. creamy, (butteryconsistency); and the third will be mucoid and having a butteryconsistency. If any doubt remains to the observer, although withreasonable care in producing the plates there should be no doubts, thecolonies can be further identified by microscopic examination. Thefirst-named colony will have organisms of 6 to 10 microns, the secondnamed colony will have organisms of 6 to 10 microns, and the third-namedcolony will have organisms of 2 to 4 microns.

The three distinct colonies noted above, are picked from the plates andplaced on separate plates made of- YM agar with 18 w/v percent of NaCland the colonies are further grown to increase the amount thereof for atleast 24 hours at mild temperatures, e.g., room temperature, but thegrowth may be continued as long as desired. However, little additionalgrowth takes place after two weeks. The procedure of growing theseparate colonies on separate YM plates is repeated until a substantialamount of the separate colonies is obtained. While no minimum amount isrequired, it is more convenient to work at least l/lOth gram of each ofthe colgional Research Lab (NRRL). The yeast medium broth consists of 3grams of yeast extract, 3 grams of malt extract, 5 grams of beefextract, 10 grams of glucose and 1,000 cc. of water, i.e. essentially YMpowder without the agar. The broth is prepared by boiling to dissolveand autoclaving to sterlize. One-third of the broth is placed in aflask. To this one-third of broth is added 1 v/v percent of Tween(Sorbitan mono-oleate) to lower the surface tension thereof. Salt (NaCl)is added to the Tween 80 containing broth and to additional flasks ofthe broth without Tween 80 to produce and 18 w/v percent solution of thesalt. One flask is inoculated with the first-named organism (the dry,flaky. white, large organisms); one flask is inoculated with thesecond-named organism (large, creamy, buttery consistency organisms);and one flask is inoculated with the third-named mucoid organism. Thefirst and secondnamed organisms are inoculated onto flasks which do notcontain the Tween 80 and the third-named organism is inoculated onto theflask which does contain Tween 80. All of the flasks are incubated atmild tempratures, e.g., room temperature for at least two weeks. Longertimes may be used as desired, but little growth takes place after fourmonths. The growth is then sepa rated from the medium of the flasks inany desired manner, such as centrifuging (where the sediment of thecentrifuging is the organisms) and the separated organisms are thencombined and are suitable for use in inoculating the Koji in the mannernoted above.

For purposes of the present specification and claims. the organismswhich are isolated as described above by either technique. are definedby the terms Maromi isolated organisms and these terms are meant todescribe the above noted isolated organisms. These terms, as used in theclaims. are intended to convey this definition. When pure organisms.obtained commercially, are used, as opposed to isolating the organisms.as noted above, it is also preferrred that at least one Saccharomycesand at least one Debaryomyces be used in combination to inoculate theKoji, although only one of the two will produce an acceptable soy sauce.The preferred species are Saccharomyces rouxii and Debaryomyceshansenii. In any case. at least 10 organisms per cc. of either thecommercially available or isolated organisms must be used to inoculatethe Maromi, preferably at least 100 organisms per cc., and especially atleast 1,000, e.g. 10,000 organisms per cc. of total liquid in preparingthe Koji, as noted above.

While the present invention has been ilustrated in terms of producingsoy sauce from Koji made from soybeans and wheat, as is apparent tothose skilled in the art, Koji may be produced with a combination ofsoybeans and other grains or cereals. For example, instead of wheat,there may be used other grains or cereals such as rice, barley, sorghum,cotton seed, corn or maize, oats or legumes such as legume seeds.Generally the proportion of soybeans, on a weight basis, to other grainsor cereal will be between to 75 percent soybeans and the remainder beingother of the cereals or grains noted above. Preferably, however, theproportion of soybeans to other cereals or grains is approximately 50percent by weight. Also, in the preferred process, one-half of thesoybeans are roasted and onehalf of the soybeans are boiled. The roastedand boiled soybeans are combined with a cereal or grain in approximatelyequal proportion, e.g., with wheat (roasted). In addition, part of thesoybeans may be replaced by other high protein content beans, such aspeas, lima beans. black-eyed peas, kidney beans, etc. However, theseother beans should not replace more than one-half of the soybeans.

Hence, the term Koji" as used in the present specifi cation and claimsembraces the above noted variation of beans, cereals and grains. Thefollowing examples will illustrate the preferred embodiments of theprocess, but it should be understood that the invention is not limitedto the following illustrative examples but is applicable to the extentof the foregoing disclosure.

EXAMPLE 1 A conventional Koji was prepared by boiling in water at 212Ffor 15 minutes 8 /1 pounds of soybeans, roasting for 1 hour in arotating oven at 250F 8 pounds of soybeans and roasting for 1 hour in arotating oven at 250F l7 /2 pounds of wheat. The boiled soybeans,

roasted soybeans and wheat were mixed until a uniform mixture wasobtained. The mixture was ground with Urschel mill until all particleshad a size of less than 10 microns. To the resulting ground mixture wasthen added 0.1 gram of dry powdered Koji culture and al' lowed toferment 5 days at 82F.

EXAMPLE 2 The Koji of Example I was placed in 167 pounds of sterilized18 w/v percent aqueous NaCl contained in a stainless steel mixing vesselfitted with a paddle stirrer and mixed until a slurry was produced. Withmixing. 13

cc. of inoculant in 18 w/v percent saline solution was added to theslurry. The inoculant contained approximately l X l0 organisms ofSaccharomyces rouxii. The mixing vessel was capped with a head platehaving a nitrogen inlet and a nitrogen outlet. Nitrogen was flushedthrough the head space of approximately 2 cu. ft. at a rate ofapproximately 10 cc/min. The slurry was maintained at between and 84F bymeans of a water jacket surrounding the mixing vessel. The aboveconditions were maintained for a total of 5 days. with a 15 minutestirring every 4 hours. After the 5 day period. the nitrogen flush wasstopped every 4 hours and replaced by an air flush for 15 minutes, withstirring during the flush and with stirring for 15 minutes after the airflush was completed and nitrogen was again flushed through the headspace. After each aeration. the nitrogen flush was resumed andcontinuously maintained. The above temperature conditions, nitrogen andair flushing schedule was continued for a total of 21 days beyond theinitial 5 day fermentation period (e.g., a total of 26 days processingtime).

Thereafter, the liquid was separated from the solids by means of aconventional filter press. The liquid had the color and aroma of soysauce. The taste was that of a good soy sauce with medium body. The soysauce was aged for two weeks in a stainless steel closed vat and theflavor developed into a very full-bodied, good tasting soy sauce.

EXAMPLE 3 The procedure of Example 2 was repeated except that theinoculant contained Debaryomyces hansenii. The resulting soy sauce had aslightly better flavor than that of Example 2.

EXAMPLE 4 The procedure of Example 2 was repeated except that theinoculant contained approximately equal amounts of Saccharomyces rouxiiand Debaryomyces hansenii. The resulting flavor was significantlysuperior to that of either Example 2 or Example 3.

EXAMPLE 5 The procedure of Examples 2, 3 and 4 were repeated except thatthe air flush used in those Examples after the first 5 day period wasinstead also used during the first 5 day period. In each case a betterflavor was obtained.

EXAMPLE 6 *YM powder is YM agar (dehydrated as disclosed in Wickerham.Tech. Bull. No. 1029. L'.S.D.A. l95l and contains yeast extract. heelextract. dextrose and agar. This is a standard commercially availablematerial Five grams of YM powder were added to l()() cc of water.sterilized at 250F for l5 seconds and designated 5 di 1 the saidinoculant having a number of organisms equiv- M di 1 d di 2 were CQ ledr 1 lF d 2t) alent to at least l.000 organisms per cc of liquid to becc. of each media was placed in separate 3 inch diameassociated with [hej in forming mummiter sterilized petri dishes (designated dish I anddish 2). The Composition of Chlim 1 wherein the ant Media 1 and 2 werestreaked with 1 year old naturally H) is admixed with koji. producedMaromi and allowed to incubate at 71F. for The Composition of Chlim 1wherein the fldmiXlure 4 days. is with maromi.

With bacteriological needles. the yeast cultures (vi- The mp ition ofClaim 1 herein the said koji sually identified) which were either flaky.dull or shiny 0T maromi has 11 Particle iz hich Will pass through wereremoved from dishes l and 2 and streaked in sep- 11 3 Us Sieve r naratepetri dishes containing the same. respective. The p i n of Claim 4wherein the particles media as described above. These dishes wereincubated wiii P1155 thmugh 11 6 Us Sieve Screen at 7lF for 4 days. Thispurifying procedure was re- 6. The composition of claim 1 wherein thecomposipeated 3 times. after which pure cultur s of flak dull tioncontains an edible salt solution and the amount of and shiny ycastcultures were obtained. The cultures 30 sail in Said Solution is from 5-percent. were identified as Saccharomyces and Debaryomyces. Theomposition of Claim 6 wherein the said salt Equal portions of the flaky.dull and shiny cultures iS flC were used in the procedure of Example 2,in the same 8. The composition Of claim 7 wherein the said solutotalamounts as in Example 2, instead of the Saccharotion ta from 5- percentof NaCl. myces rouxii. The produced soy sauce had an excellent 35 TheComposition of Claim 8 wherein the Said lufl d f ll b d tion containsapproximately 18 w/v percent of NaCl.

10. The composition of claim 1 wherein the koji or EXAMPLES 7 i i maromicontains a grain or cereal.

The procedure of Example 2 was repeated except 11. The composition Ofclaim 10 wherein the grain 01' that the conditions and materials. asshown in Table l, Cereal i5 fl were used The results are noted in [uble12- The composition Of claim wherein the said koji contains from 25-75percent by weight of soybeans and from 75-25 percent by weight of wheat.EXAMPLE I2 13. The composition of claim 12 wherein approxi- Theprocedure of Example 2 was repeated except mately one-half ofthesoybeans have been boiled, onethat no aeration was performed during theprocess. The half of the soybeans have been roasted and the wheatresulting flavor was inferior to the flavor of Example 2. has beenroasted. but was still the characteristic soy sauce flavor. 14. Thecomposition of claim I wherein the maromi As can be seen from theforegoing disclosure, soy contains up to solids. sauce can be producedwith very short total fermenta- 40 15. The composition of claim 1wherein the yeast are tion times. e.g.. in less than 2 months andespecially in both a Saccharomyces and a Debaryomyces. about 30 days.This. of course. results in a significant 16. The composition ofclaim 15wherein the Sacchasaving in time and money over the conventionalperiromyces is S. rouxii and the Debaryomyces is D. hanods of 1 year to18 months. senii.

TABLE I 12 of koji or maromi with which is additionally admixed aninoculant having as the sole essential organisms of the said inoculantat least one yeast selected from the group consisting essentially ofSaccharomyces. Debaryomyces. Maromi isolated yeasts and mixturesthereof.

First Fermental Temp.

Second Fermentation Temp.

Example Koji lnoculant And Time F. and Time, Days Flavor 7 2 lbs.roasted lXlO Sacch. F, 7 days F, l4 days Good flavor medium so beansrouxii body 3 bs. boiled soybeans and 30 lbs. roasted wheat 3 lXlOSacch. Mild flavor light rouxii bod 9 1X10 Sacch. 83F, 5 days 83F. 21days Very distinct flavor rouxii and full body 10 20 lbs. boiled lXlO'"Sacch. 84F. 7 days 84F. 25 days Strong flavor soybeans rouxii full body5 lbs. roasted soybeans l2 lbs. roasted wheat II What is claimed is: 1.An anaerobic to mildly aerobic fermentable composition for producing soysauce comprising a mixture lO5F, 7 days slightly acid taste 4 full body17. The composition of claim 1 wherein the said yeast are maromiisolated organisms.

1. AN ANEROBIC TO MILDLY AEROBIC FERMENTABLE COMPOSITION FOR PRODUCINGSOY SAUCE COMPRISING A MIXTURE OF KOJI OR MAROMI WITH WHICH ISADDITIONALLY ADMIXED AN INOCULANT HAVING AS THE SOLE ESSENTIAL ORGANISMSOF THE SAID INOCULANT AT LEAST ONE YEAST SELECTED FROM THE GROUPCONSISTING ESSENTIALLY OF SACCHAROMYCES, DEBARYOMYCES, MAROMI ISOLATEDYEASTS AND MIXTURES THEREOF, THE SAID INOCULANT HAVING A NUMBER OFORGANISMS EQUIVALENT TO AT LEAST 1,000 ORGANISMS PER CC OF LIQUID TO BEASSOCIATED WITH THE KOJI IN FORMING MAROMI.
 2. The composition of claim1 wherein the inoculant is admixed with koji.
 3. The composition ofclaim 1 wherein the admixture is with maromi.
 4. The composition ofclaim 1 wherein the said koji or maromi has a particle size which willpass through a No. 3 US sieve screen.
 5. The composition of claim 4wherein the particles will pass through a No. 6 US sieve screen.
 6. Thecomposition of claim 1 wherein the composition contains an edible saltsolution and the amount of salt in said solution is from 5-30 w/vpercent.
 7. The composition of claim 6 wherein the said salt is NaCl. 8.The composition of claim 7 wherein the said solution contains from 15-20w/v percent of NaCl.
 9. The composition of claim 8 wherein the saidsolution contains approximately 18 w/v percent of NaCl.
 10. Thecomposition of claim 1 wherein the koji or maromi contains a grain orcereal.
 11. The composition of claim 10 wherein the grain or cereal iswheat.
 12. The composition of claim 11 wherein the said koji containsfrom 25-75 percent by weight of soybeans and from 75-25 percent byweight of wheat.
 13. The composition of claim 12 wherein approximatelyone-half of the soybeans have been boiled, one-half of the soybeans havebeen roasted and the wheat has been roasted.
 14. The composition ofclaim 1 wherein the maromi contains up to 40% solids.
 15. Thecomposition of claim 1 wherein the yeast are both a Saccharomyces and aDebaryomyces.
 16. The composition of claim 15 wherein the Saccharomycesis S. rouxii and the Debaryomyces is D. hansenii.
 17. The composition ofclaim 1 wherein the said yeast are maromi isolated organisms.